Altered purine and pyrimidine metabolism in erythrocytes with purine nucleoside phosphorylase deficiency

Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07% of normal purine nucleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency—that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis—could not be supported by observations in erythrocytes from both enzyme-deficient families.This work was supported by U.S. Public Health Service Grant AM 19674 and 5 M01 RR 42 and by a Grant-In-Aid from American Heart Association (77-849) and with funds contributed in part by the Michigan Heart Association. N.L.E. is a Rheumatology Fellow from the Rackman Arthritis Research Unit supported by Training Grant USPHS AM 07080.     

Hypoxanthine-guanine phosphoribosyltransferase: Mosaicism in the peripheral erythrocytes of a heterozygote for a normal and a mutant enzyme

A mutant form of erythrocyte hypoxanthine-guanine phosphoribosyltransferase with an abnormal isoenzyme pattern was found in a patient with a partial enzyme deficiency and X-linked gout. This abnormal pattern was a marker for the mutant enzyme in hemolysate from the heterozygote for the enzyme deficiency.These studies were generously supported by the Medical Research Council of Canada (MRC # MA4758) and the Canadian Arthritis and Rheumatism Society.     

Rapid prenatal diagnosis of HG-PRT deficiency using ultra-microchemical methods

Summary A previously developed simple ultramicromethod has been used for the rapid prenatal diagnosis of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) deficiency. The method is based on the incubation of small numbers of visually selected, lyophilized fibroblasts (in the present study five cells per incubation) with radioactive substrate in an end volume of 0.3 l. Fibroblasts derived from the amniotic fluid of a 15-week male fetus in a heterozygote for the X-linked Lesch-Nyhan syndrome showed a severe degree of HG-PRT deficiency. In total 50 fibroblasts were used. The diagnosis was confirmed upon termination of the pregnancy by the demonstration of HG-PRT deficiency in fetal erythrocytes and cultured skin fibroblasts.HG-PRT: hypoxanthine-guanine phosphoribosyl transferase     

X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: Detection of heterozygotes by selective medium

Skin fibroblasts from five unrelated males with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency and from their families have been exposed to medium containing 6-thioguanine. This purine analogue selects against cells with normal hypoxanthine-guanine phosphoribosyl transferase activity and therefore permits detection of mutant cells in heterozygous populations. The results of these studies are compared to those obtained by autoradiography of single-cell clones of skin fibroblasts from the same subjects. In each case, the results of the selective method are similar to those obtained by clonal analysis. The use of selective medium therefore provides a sensitive means to detect heterozygosity at this locus and may provide a general method to select cells with X-linked markers from heterozygous populations.This work was supported by grants from the U.S.P.H.S. (#HD 00486), The Joseph P. Kennedy, Jr., Memorial Fluid Research Fund, and the National Foundation for Neuromuscular Diseases, Inc.     

Activities of X-linked enzymes in spermatocytes of mice rendered sterile by chromosomal alterations

Spermatocytes in the late prophase of first meiotic division isolated from sterile males retain higher activities for three X-linked enzyme⁵, phosphoglycerate kinase (PGK)-1, glucose-6-phosphate dehydrogenase (G6PD), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) than those of fertile males. The sterilty of the male is presumed to be owing to the rearrangement of X-chromosome material and the possibility of abnormal meiotic X-chromosome inactivation is discussed.     

Hypoxanthine-guanine phosphoribosyl transferase with altered substrate affinity in mutant mouse lymphoma cells

Cells with altered hypoxanthine-guanine phosphoribosyl transferase (HPRT) (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) have been selected. Compared to wild type, mutant enzyme has a reduced affinity for the substrate phosphoribosyl pyrophosphate and is more labile to heat inactivation. Mutant cells are resistant to 6-thioguanine at 33-39 degrees C and sensitive to hypoxanthine-aminopterin-thymidine at 37-39 degrees C, but not at 33 degrees C. We hypothesize that a single structural mutation of HPRT can explain these results.     

Communication between normal and enzyme deficient cells in tissue culture

Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoriboxyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly (L-lysine), lysolecithin and amphotericin B seem to inhibit the entry of the enzymes of their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10--20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoribosyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly(L-lysine, lysolecithin and amphotericin B seem to inhibit the entry of the enzymes or their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10–20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

Genetic studies of human erythrocyte inosine triphosphatase

The high concentrations of inosine triphosphate in human erythrocytes of some subjects has been related to a deficiency in intracellular inosine triphosphatase. Evidence has been presented for genetic transmission of this enzyme and for the existence of a homozygous-heterozygous relationship. Pedigree studies of individuals with erythrocyte ITPase deficiency suggest a Mendelian autosomal trait.This investigation was partly supported by PHS Research Grant No. Am-11116 from the National Institute of Arthritis and Metabolic Disorders.     

Nature of the heterozygote blood catalase in a hypocatalasemic mouse mutant

The blood catalase of a hypocatalasemic mouse mutant has been compared with that of the wild-type (normal) animal and with that of the heterozygote. Comparison is on the basis of stability to heat and to urea. Electrophoretic evidence is of no value, because all forms tested show the same mobility. Because the heterozygote heat and urea inactivation curves differ from those of the two parental forms, and because the curves are smoothly S-shaped, with no shoulders or other irregularities, it is suggested that this heterozygote produces only a single molecular form of the enzyme.Work supported under the auspices of the U.S. Atomic Energy Commission.     

The mode of genetic transmission of a gouty family with increased phosphoribosylpyrophosphate synthetase activity

Summary The mode of genetic transmission of gout and increased activity of phosphoribosylpyrophosphate synthetase (PRPPS) was studied in one family. Among 15 members of Family F, two male members had gout and had PRPPS activity of erythrocyte lysates three times higher than normal subjects. Five female members had activity 2.5 times higher than normal. The difference between the activities of male and female affected members was statistically significant (P<0.05). To examine the genetic trait of this abnormal PRPPS, the incorporation of ³H-adenine into erythrocytes or lymphocytes was studied using autoradiography. The number of grains which show the uptake of labeled adenine into cells revealed a normal distribution pattern in two normal persons and in two male patients, and a mixed pattern of the two cell populations in two female affected members. These results suggested mosaicism in female members and X-linked dominant transmission of this trait. Thermal inactivation of PRPPS of an affected female was intermediate between that from a normal subject and that from the affected males. This result showed the heterogeneity of the PRPPS from the hemolysate of an affected famale. The genotype of PRPPS on the X-chromosome was assumed and the lod score between PRPPS and Xg was also estimated. From these findings and electrophoretical study, it was suggested that the abnormal enzyme was a mutant enzyme transmitted in an X-linked dominant trait, and that the mutation occurred on the structural gene of the PRPPS.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier.

Clonal myogenic cell cultures were established from a potential heterozygote for a mutant Duchenne muscular dystrophy (DMD) gene who was also heterozygous for isozymes of the X-linked enzyme glucose-6-phosphate dehydrogenase. Previous tissue culture studies of this muscle donor demonstrated equal proliferative capacity of myoblasts that had lyonized either the paternal or maternal X-chromosome, indicating that mutation of the DMD gene does not affect growth of myoblasts. If this muscle donor were a gonadal mosaic, this conclusion would be incorrect. In the present study, only those myogenic colonies expressing the glucose-6-phosphate dehydrogenase-A isozyme were found to express dystrophin, indicating that this woman was indeed a heterozygote for DMD. By documenting dystrophin deficiency in a specific population of myogenic cells from this woman, we verify our previous conclusion regarding the normal proliferative capacity of DMD myoblasts. Somatic cell testing of dystrophin expression may offer an alternative to established genetic carrier tests for those women in whom deletions of the DMD are not detectable, whose pedigree structure does not permit linkage analysis, or in whom standard phenotypic analyses are ambiguous.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.     

X-chromosome inactivation and selection in somatic cells.

X-Chromosome inactivation leads to the formation of mosaic cell populations in the somatic cells of mammalian females. Cells have either a maternal or paternal X-chromosome active. If an individual is heterozygous for a cell autonomous X-linked trait, then a set of built-in cellular markers is provided for the investigation of various developmental phenomena, including selection. In the absence of somatic cell selection the tissues of an X-linked heterozygote should all be mosaic and should have a mosaic composition approaching 1:1. If somatic cell selection is occurring, it should be detectable by a significance shift from the random expected 1:1 mosaicism. The system is effective at detecting selection acting on X-linked loci and on newly arisen somatic autosomal variants, and several examples of somatic cell selection are described. However, it is concluded that somatic cell selection, as described above, is not a normal aspect of ontogeny.     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

Metabolic cooperation studied by a quantitative enzyme assay of single cells

A new method making use of a radiochemical enzyme assay at the single cell level is presented to investigate metabolic cooperation, a widely studied form of cellular communication. In this case metabolic cooperation between normal human fibroblasts and fibroblasts derived from a patient deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase has been studied.A mixture of an equal number of both cell types was cultured in close physical contact and after trypsinisation, replating and culturing the cells for several hours in a high dilution, quantitative enzyme measurements with individual cells isolated from the mixture were carried out. From the distribution curve of the enzyme activities of the individual cells the conclusion could be drawn that a macromolecule, either the enzyme itself or DNA or mRNA, coding for that enzyme, is transferred from normal to mutant cells.     

Comparison of the properties of histidine ammonia-lyase in normal and histidinemic mutant mice

The histidinemic (his/his) mutant mouse shows greatly reduced skin and liver histidine:ammonia-lyase (HAL; EC 4.3.1.3) activity compared with normal mice. Liver HAL activity in the mutant is heat and salt labile and is inhibited at high substrate concentrations. Two HAL components have been identified in the normal mouse liver, a minor component with properties similar to those of HAL of the mutant mouse and a major component which is heat and salt stable and insensitive to substrate inhibition. Immunotitration with anti-HAL antibody shows that the livers of mutant mice contain no detectable antigenically cross-reacting HAL protein. It is concluded, therefore, that the his allele is a null allele at a structural or regulatory locus for the major HAL enzyme and maps close to the HAL-regulatory locus Hsd and that the low residual HAL activity in the mutant is due to another enzyme.This work was supported by an MRC Project Grant to H.K. and USPHS Grant GH21002 to S.M.A.     

Deficiency of AMP deaminase in erythrocytes

Summary Six individuals with complete deficiency of erythrocyte AMP deaminase have been discovered. They are all healthy and have no hematological disorders. The deficiency is only in isozyme E, which is the erythrocyte type isozyme, and is inherited as an autosomal recessive trait. The frequency of the mutant gene is surprisingly high, one heterozygote in about 30 of the population in Japan, Seoul, and Taipei. The ATP level is approximately 50% higher in AMP-deficient erythrocytes compared to that of control cells. Degradation of adenine nucleotide is slower in the deficient erythrocytes than in the control erythrocytes.